This information is current as
of June 18, 2017.
Hsp70 Translocates into the Plasma
Membrane after Stress and Is Released into
the Extracellular Environment in a
Membrane-Associated Form that Activates
Macrophages
Virginia L. Vega, Monica Rodríguez-Silva, Tiffany Frey,
Mathias Gehrmann, Juan Carlos Diaz, Claudia Steinem,
Gabriele Multhoff, Nelson Arispe and Antonio De Maio
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2008 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2008; 180:4299-4307; ;
doi: 10.4049/jimmunol.180.6.4299
http://www.jimmunol.org/content/180/6/4299
The Journal of Immunology
Hsp70 Translocates into the Plasma Membrane after Stress
and Is Released into the Extracellular Environment in a
Membrane-Associated Form that Activates Macrophages1
Virginia L. Vega,* Monica Rodrı́guez-Silva,* Tiffany Frey,† Mathias Gehrmann,‡
Juan Carlos Diaz,§ Claudia Steinem,¶ Gabriele Multhoff,‡ Nelson Arispe,§
and Antonio De Maio2*†
E
xpression of heat shock proteins (hsps)3 is markedly increased as part of the response to an array of stressors.
These proteins participate in the refolding of denatured
polypeptides that become damaged as a consequence of an insult.
During nonstress conditions, hsps participate in the folding of nascent polypeptides and the stabilization of receptors and signal
transduction molecules (1). Although hsps are localized intracellularly, they have also been found outside the cell, particularly
after various pathological conditions (2– 4). Extracellular hsps,
specifically Hsp70, have been reported to activate macrophages,
dendritic cells, and NK cells by a receptor-mediated process (5–
10). An important question that remains unanswered is how does
Hsp70, which does not have a consensual secretory signal, reach
the extracellular environment? Initially, it was thought that Hsp70
was released from necrotic cells after injury (11). However, secretion of Hsp70 also occurs in the absence of cell death (12). Consequently, an active, nonclassical secretory pathway may be involved. We have previously shown that both Hsp70 and heat shock
cognate 70 (Hsc70) interact with artificial membranes (13). Moreover, these proteins displayed a particular specificity for phosphatidylserine (PS), a phospholipid that is normally present in the
cytosolic side of cellular membranes (14). In the present study, we
investigate whether the interaction of Hsp70 with membranes acts
as a platform for its release into the extracellular environment.
Materials and Methods
Materials
*Department of Surgery, University of California San Diego, La Jolla, CA 92093;
†
Graduate Program Cellular and Molecular Medicine, Johns Hopkins University, Baltimore, MD 21205; ‡Klinikum Rechts der Isar, Technische Universität München, and
Helmholtz Center Munich Clinical Cooperation Group Innate Immunity, Munich,
Germany; §Uniformed Services University of the Health Sciences, Bethesda, MD
20814; and ¶Institute for Organic and Biomolecular Chemistry, University of Göttingen, Göttingen, Germany
Received for publication October 5, 2007. Accepted for publication January 11, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grant GM 50878 (to
A.D.M.) and Deutsche Forschungsgemeinschaft Grant MU 1238 7/2 (to G.M.).
2
Address correspondence and reprint requests to Dr. Antonio De Maio, University of
California San Diego Department of Surgery, 9500 Gilman Drive, Number 0739, La
Jolla, CA 92093. E-mail address: [email protected]
3
Abbreviations used in this paper: hsp, heat shock protein; CTX, cholera toxin;
DAPI, 4⬘,6⬘-diamidino-2-phenlyindole; DRM, detergent-resistant membrane; ECM,
extracellular membrane; GA, geldanamycin; GM1, monosialoganglioside; HS, heat
shock; Hsc70, heat shock cognate 70; M␤CD, methyl-b-cyclodextrin; PFA, paraformaldehyde; POPE, 1-palmitoyl-2-oleoyl-phosphatidylethanolamine; POPS, 1-palmitoyl-2-oleoyl-phosphatidylserine; PS, phosphatidylserine; SF, serum free; sulfo-NHSbiotin, N-hydroxysulfosuccinimidobiotin; WT, wild type; YFP, yellow fluorescent
protein.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
www.jimmunol.org
Cell lines were obtained from American Type Culture Collection. Alexa
Fluor 532-conjugated cholera toxin (CTX) subunit B, Alexa Fluor 594conjugated transferrin, and Amplex Red cholesterol assay kits were purchased from Invitrogen Life Technologies. Ab against the C-terminal region of human Hsp70, cmHsp70.1, was obtained from Multimmune. Abs
against the N-terminal region of Hsp70 (SPA-810), Hsc70 (SPA-815),
Hsp40 (SPA-450), Hsp27 (SPA-800), and Hsp90 (SPA-830) were purchased from StressGen Bioreagents. HRP-conjugated streptavidin was purchased from Invitrogen Life Technologies. HRP-conjugated CTX, methyl␤-cyclodextrin, and mouse monoclonal anti-actin Ab were purchased from
Sigma-Aldrich. EZ-link N-hydroxysulfosuccinimidobiotin (sulfo-NHS-biotin) reagent was purchased from Pierce. Palmitoyl-oleoyl-phosphatidyl
serine and palmitoyl-oleoyl-phosphatidylcholine were obtained from
Avanti Polar Lipids. TNF-␣ ELISA was obtained from BioSource International. FuGENE transfection reagent was purchased from Roche Applied
Science.
Immunostaining
Cells (1 ⫻ 106) were grown on a sterile glass cover slide. Nonspecific
binding was blocked by incubation with serum (20%) from the host of
secondary Ab and 0.2% Tween 20 in PBS (0.5 h at 25°C). Cells were
incubated with primary Abs (1/200 dilution) for 1 h at 4°C, extensively
washed with PBS, and incubated with secondary Abs (1/1000 dilution) for
0.5 h at 4°C. When indicated, cells were fixed with 4% paraformaldehyde
(PFA) (10 min at 25°C) and permeabilized with cold acetone (15 s). In
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Heat shock proteins (hsps) are intracellular chaperones that play a key role in the recovery from stress. Hsp70, the major
stress-induced hsp, has been found in the extracellular medium and is capable of activating immune cells. The mechanism involved
in Hsp70 release is controversial because this protein does not present a consensual secretory signal. In this study, we have shown
that Hsp70 integrates into artificial lipid bilayer openings of ion conductance pathways. In addition, this protein was found
inserted into the plasma membrane of cells after stress. Hsp70 was released into the extracellular environment in a membraneassociated form, sharing the characteristics of this protein in the plasma membrane. Extracellular membranes containing Hsp70
were at least 260-fold more effective than free recombinant protein in inducing TNF-␣ production as an indicator of macrophage
activation. These observations suggest that Hsp70 translocates into the plasma membrane after stress and is released within
membranous structures from intact cells, which could act as a danger signal to activate the immune system. The Journal of
Immunology, 2008, 180: 4299 – 4307.
4300
EXPORT OF MEMBRANE-BOUND HSP70
some cases, immunostained cells were incubated with 1% Triton X-100 in
PBS for 20 min on ice. Nuclei were stained with 4⬘,6⬘-diamido-2-phenylindole (DAPI) hydrochloride (15 s at 25°C), and cells were visualized using
a fluorescent microscope.
Recording of Hsp70 ion channel activity using a planar lipid
bilayer
The experimental chamber consisted of two compartments separated by a
thin Teflon film with a hole of ⬃100- to 120-␮m in diameter. Planar lipid
bilayers were formed by applying a suspension of 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE) and 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS) (1:1, 50 mg/ml, each in n-decane) to the hole in the Teflon
film. The ionic solutions contained asymmetrical concentrations of KCl
(200 mM cis and 50 mM trans), and 0.5 mM CaCl2, 1 mM MgCl2, and 5
mM potassium-HEPES (pH 7). Hsp70 was incorporated into the lipid bilayer from POPS liposomes bearing the peptide and added to the cis compartment in small aliquots. To prepare Hsp70 liposomes, POPS dissolved
in CHCl3 (10 mg/ml) was air dried and resuspended in 1 M potassium
aspartate to a final concentration of 5 mg POPS/ml. The resulting mixture
was bath sonicated for 5 min. Aliquots of the stock solution of Hsp70 were
added to the liposome suspension, and the combination was sonicated for
an additional period of 2 min. Recombinant Hsp70 used in this study was
a special order from StressGen Bioreagents that was dialyzed extensively
to eliminate ATP and salts. Incorporation of Hsp70 occurred directly from
the solution by spontaneous fusion of the proteoliposomes with the lipid
bilayer. Channel currents associated with the incorporation of Hsp70 were
recorded using a patch-clamp amplifier and stored on computer disk memory. Off-line analysis of the recorded Hsp70 channel activity was conducted using the software package pClamp.
Detergent-resistant membrane (DRM) isolation
HepG2 cells (90% confluent, 10-cm culture dish) were harvested in 1 ml of
cold TNE buffer (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA,
4 mg/ml trypsin inhibitor, 1 mg/ml benzamide, 5 ␮mol/ml leupeptin, 200
␮M sodium vanadate, 100 nM okadaic acid, and 1 mg/ml PMSF) containing 1% Triton X-100. Cell lyses were incubated at 4°C for 0.5 h and an
equal volume of 85% sucrose in TNE was added. The mixture was placed
at the bottom of a centrifuge tube and overlaid with 4 ml of 35% sucrose
and 1 ml of 5% sucrose followed by 4.5 ml of TNE buffer and centrifuged
at 200,000 ⫻ g for 18 h at 4°C (15). After centrifugation, 3.5 ml were
removed from the top of the tube and 13 fractions (615 ␮l each) were
collected from the top of the gradient. Each fraction (535 ␮l) was precipitated with 10% TCA (2 h at 4°C) and centrifuged at 15,600 ⫻ g (10 min
at 4°C). Samples were washed twice with cold acetone, resuspended in 1⫻
loading buffer, boiled, and separated by SDS-PAGE. The rest of each fraction (80 ␮l) was used for the detection of monosialoganglioside (GM1) slot
blots using HRP-conjugated cholera toxin.
Extracellular membrane (ECM) isolation
HepG2 cells, (90% confluent, 150-cm culture dish) were heat shock (HS)
or not in serum-free (SF) medium and allowed to recover for different
lengths of time at 37°C. The ECM was collected and centrifuged at
1,500 ⫻ g for 10 min. The supernatant was centrifuged at 10,000 ⫻ g (0.5
h at 4°C) followed by ultracentrifugation at 100,000 ⫻ g (1 h at 4°C). The
pellet was resuspended in 1 ml of supernatants and ultracentrifuged again
at 100,000 ⫻ g (1 h at 4°C). The resulting pellet (ECM) was resuspended
in loading buffer 1⫻ or PBS. This protocol has been previously used to
isolate exosomes from cells (16, 17).
Cell surface and ECM biotinylation
HepG2 cells (90% confluent) were washed twice with cold PBS and incubated with sulfo-NHS-biotin in PBS (0.2 mg/ml) for 0.5 h at 4°C (18).
Free reagent was removed by two washes with cold PBS and cells were
lysed. Isolated ECM were incubated with sulfo-NHS-biotin in PBS (1.6
mg/ml for 2 h at 4°C) and centrifuged at 100,000 ⫻ g (60 min at 4°C). The
detection of biotinylated proteins was performed using HRP-conjugated
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FIGURE 1. Incorporation of Hsp70 into a planar
lipid bilayer opens a stable ion conductance pathway.
Proteoliposomes made of purified human recombinant
Hsp70 and POPS were added to one of the sides of the
experimental chamber, and the current activity at the
planar lipid bilayer was measured. The planar lipid bilayer, made of a suspension of POPS/POPE, was separating ionic solutions of asymmetrical concentrations of
KCl (200 mM cis and 50 mM trans). A, At low time
resolutions, the Hsp70 channel activity shows stable
trains of brief current spikes, indicating brief multiple
channel conductance changes. B, At a more expanded
time scale, the current traces show the long duration
channel openings, indicating channel stabilization into
one preferred conductance state. C, The channel activity
made of discrete events shows frequent switching between well-defined, long-lasting current levels. The dotted lines on the traces indicate the best-defined current
levels and their values, which were obtained from the
current amplitude histogram (D). The current amplitude
histogram shows the frequency of unitary opening
events lasting ⬎2 milliseconds. Marquardt least square
fitting of this histogram indicates that the current values
distributed mainly into five Gaussians. The values corresponding to each current peak in the histogram are: a,
0.533 ⫾ 0.007; b, 1.068 ⫾ 0.046; c, 1.677 ⫾ 0.085; d,
2.496 ⫾ 0.006; and e, 3.697 ⫾ 0.045. Data are from a
representative experiment of ⱖ42 determinations.
The Journal of Immunology
4301
streptavidin (1/5000 at 3 h) after SDS-PAGE and transferring onto nylon
membranes.
Results
Hsp70 forms stable multiconductance ion channels in planar
lipid bilayers
Hsp70 is present on the plasma membrane after stress
The preceding observations suggest that Hsp70 is capable of interacting with lipids within membranes. We further investigated
whether Hsp70 is present in cellular membranes. HepG2 cells,
which were subjected to a 43°C HS and recovered for 7 h at 37°C,
were incubated with Abs that recognize different Hsp70 epitopes.
One of these Abs (cmHsp70.1) is directed against a peptide localized in the C terminus of Hsp70, whereas the other Ab (SPA-810)
recognizes the N terminus of the molecule. The presence of Hsp70
on the plasma membrane was observed after staining with FITCconjugated cmHsp70.1 Ab at 4°C in nonpermeabilized, nonfixed
cells (Fig. 2, A and C). Under the same conditions, SPA-810 Ab
failed to reveal the presence of Hsp70 (Fig. 2A). In contrast, both
Abs showed a similar staining pattern in acetone-permeabilized
cells (Fig. 2A). These observations suggest that Hsp70 is inserted
into the plasma membrane with the C terminus exposed outside of
the cell. Treatment of HepG2 cells with geldanamycin (GA), an
inhibitor of Hsp90 that triggers the induction of Hsp70 (20, 21),
and destabilizes membranes, also resulted in cell surface detection
of Hsp70 by cmHsp70.1 Ab (Fig. 2, B and C), suggesting that the
translocation of Hsp70 to the membrane is not an exclusive effect
of HS. Hsc70, the constitutive form of the Hsp70 family, was not
detected on the cell surface of nonstressed or stressed cells (data
not shown). The presence of Hsp70 on the cell surface, which was
detected by FITC-conjugated cmHsp70.1 Ab (C terminus of the
molecule), was not affected by pretreatment of the cells (10 min)
with low pH (5.0) or high salt buffer, suggesting that the protein is
FIGURE 2. Hsp70 is present on the surface of stressed cells. A,
HepG2 cells were HS (43°C for 1.5 h), recovered for 7 h at 37°C, and
incubated with Abs against Hsp70 C-terminal (cmHsp70.1) or N-terminal (SPA-810) Ab at 4°C. Nonbound Ab was washed off and cells
were fixed with PFA (top panel). The inset shows the detection of
Hsp70 on the cell surface. Heat shocked cells were fixed with PFA,
permeabilized with cold acetone, and stained for Hsp70 using either Cor N-terminal Ab (bottom panel). Nuclei (N) were stained with DAPI.
B, Cells were also treated with GA (1 ␮g/ml) for 8 h or not (control) and
incubated with FITC-conjugated cmHsp70.1 (C-terminal) Ab at 4°C as
described above. C, A comparison between surface staining using cmHsp70.1 Ab at 4°C and phase contrast for cells after HS or GA treatment. D, Detection of Hsp70 or Hsc70 by Western blotting on total cell
homogenates (H) or membrane fractions (M) obtained from unstressed
(C) or thermally stressed (HS) cells.
not bound to the plasma membrane via another surface protein
(data not shown). Hsp70 was detected by Western blotting in isolated total cellular membrane fractions from stressed cells, confirming the interaction of Hsp70 with membranes. In contrast,
Hsc70 was not detected in the membrane fraction, suggesting
that the presence of Hsp70 is not due to cytosolic contamination
(Fig. 2D).
Hsp70 on membranes is present in Triton X-100 in soluble form
Prior studies have shown that Hsp70 is present within DRM fractions. Consequently, we investigated whether Hsp70 on membranes could be present in this fraction. DRM were isolated after
Triton X-100 treatment of HepG2 cells following HS by sucrose
gradient centrifugation. Hsp70 was detected in DRM fractions,
which were identified by the presence of GM1, as early as 4 h into
the recovery time (37° C) following HS. Other hsps, such as
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We investigated the interaction of Hsp70 with membranes using
planar lipid bilayers. Proteoliposomes made of PS and human recombinant Hsp70 were added to ionic solutions that were separated by the artificial lipid bilayer in an experimental chamber. The
incorporation of Hsp70 into the lipid bilayer occurred by spontaneous fusion of the proteoliposome with the artificial membrane.
Soon after the addition of Hsp70 liposomes, an electrical current
activity was observed across the voltage-clamped bilayer, indicating a flow of charges across a newly formed ion pathway. This
current activity was observed in all cases where the incorporation
of Hsp70 into the lipid bilayer was attempted (n ⫽ 42) and showed
high stability. The direction of the current recorded under a transbilayer chemical gradient, at zero transbilayer potential, indicated
that Hsp70 preferentially permits the flow of cations. Representative recordings illustrate two different patterns of electrical activity
within the same channel performance: a stable train of swift current spikes that indicate brief, multiple channel conductance
changes (Fig. 1A) and an extended duration of opening activity that
indicates channel stabilization into one preferred conductance state
(Fig. 1B). At a long, expanded time interval and a constant membrane potential, Hsp70 channel activity showed frequent changes
between well-defined and long-lasting current levels (Fig. 1, C and
D). Analysis of the current amplitudes at different membrane potentials showed that Hsp70 channels displayed a range of measurable, stable conductance between 3 and 60 pS. Recombinant
Hsp70 used in this study was extensively dialyzed to eliminate any
possible low m.w. contaminants. In addition, the recombinant protein displayed a single band after SDS-PAGE. Neither Hsp90 nor
LPS added to the lipid bilayer displayed any channel activity,
which is consistent with a prior report (19).
4302
EXPORT OF MEMBRANE-BOUND HSP70
FIGURE 3. Hsp70 is present in DRM fractions depending on the presence of cholesterol. A, HepG2 cells were maintained at 37°C (C) or HS
(43°C for 1.5 h), recovered (37°C for 4 h), and lysed in TNE buffer containing 1% Triton X-100. DRM fractions were isolated as described in
Materials and Methods. The presence of Hsp70, Hsc70, Hsp90, Hsp27,
and actin within DRM fraction was detected by Western blotting (left
panel), whereas the levels of GM1 were measured by slot blotting using
HRP-conjugated CTX (right panel). B, Cells, controls or after HS and
recovery, were treated with M␤CD (10 mM for 30 min at 37°C) and
DRM were analyzed as described above for the presence of Hsp70 or
Hsc70. These are representative experiments of at least three independent determinations.
Triton X-100-treated cells (Fig. 5A). Similarly, the signal corresponding to Alexa Fluor 532-conjugated CTX, which binds to
GM1, was resistant to Triton X-100 treatment (Fig. 5A). In contrast, the presence of transferrin receptor was significantly decreased after incubation with Triton X-100 (Fig. 5A). To determine
whether Hsp70 and GM1 are located on the same membrane domain, cells after HS and recovery were incubated with Alexa Fluor
532-conjugated CTX at 4°C, fixed with PFA (4%), permeabilized
with cold acetone, and incubated with FITC-conjugated cmHsp70.1 Ab. Cells were then treated with Triton X-100 (1% for 20
min at 4°C). Under these conditions, colocalization of Hsp70 and
GM1 could be easily demonstrated (Fig. 5B).
Hsp70 can be detected in extracellular membranes
Hsc70, Hsp90, Hsp27, and Hsp40, were not detected in DRM fractions. Actin was only observed within DRM fractions after 4 h of
recovery from HS (Fig. 3A). Treatment of cells after HS with
methyl-␤-cyclodextrin (M␤CD), which removes cellular cholesterol without compromising cellular viability, disrupted the interaction of Hsp70 with DRM fractions (Fig. 3B). Under these
conditions, the presence of GM1 within DRM fractions was not
affected. The previous observations were extended to other cell
types. The presence of surface Hsp70 was also revealed on
K562 and Jurkat cells analyzed by fluorocytometry using FITCconjugated cmHsp70.1 Ab. Treatment of these cells with
M␤CD reduced considerably the detection of Hsp70 on the cell
surface (Fig. 4). These observations altogether suggest that the
interaction of Hsp70 with membranes is enhanced by the presence of cholesterol.
To further investigate the presence of Hsp70 on the cell surface,
HepG2 cells after HS and recovery were incubated with FITCconjugated cmHsp70.1 Ab, Alexa Fluor 532-conjugated CTX, or
Alexa Fluor 592-conjugated transferrin at 4°C, fixed with PFA
(4%) after ligand binding and treated or not with Triton X-100 (1%
for 20 min at 4°C). The signal for Hsp70 was still preserved on
We investigated whether Hsp70 could be present in the extracellular environment in a membrane-bound form. HepG2 cells were
subjected to HS (43°C, for 1.5 h) in SF medium followed by recovery at 37°C for different lengths of time. The ECM was collected and subjected to differential centrifugation. Hsp70 was only
detected in the high-speed centrifugation pellet fraction after 24 h
of recovery following HS by Western blotting. Hsp70 was not
observed in similar high-speed centrifugation fractions obtained
from nonstressed (control) cells (Fig. 6A). This high-speed centrifugation fraction, which we have named extracellular membranes or ECM, has been referred to as exosomes in other studies
(17, 22). ECM derived from both control and HS cells were positive for acetyl cholinesterase activity and contained Rab4. Actin
was detected only in fractions derived from cells subjected to HS
followed by 24 h of recovery (Fig. 6B). In contrast, other hsps,
such as Hsc70, Hsp90, or Hsp27, were not detected in ECM (Fig.
6B). ECM contain cholesterol and GM1 and can be stained by
FM4 – 64 reagent, which interacts with phospholipids. Hsp70
within ECM was resistant to Triton X-100 solubilization and
Na2CO3, extraction suggesting that Hsp70 is inserted into the
membrane (Fig. 6C). To demonstrate that solubilization by Triton
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FIGURE 4. Cell surface presence of Hsp70 is affect by M␤CD treatment. K562 or Jurkat cells (0.5 106 cells/ml) were treated or not with
M␤CD (1 mM for 6 h at 37°C), washed with PBS, and stained with
FITC-conjugated cmHsp70.1 Ab (Multimmune). Labeled cells were analyzed using a FACSCalibur apparatus. Top panel, representative histograms of mean fluorescence intensity, cmHsp70.1 (thick line), and
isotype-matched control Ab (light line); dashed lines correspond to
M␤CD treatment; Bottom panel, Signal intensity quantification for cell
surface Hsp70 (n ⫽ 4). ⴱ, p ⬍ 0.05 vs untreated cells.
The Journal of Immunology
X-100 indeed removes some proteins from ECM, they were labeled with sulfo-NHS-biotin after isolation. Treatment of these
labeled membranes with Triton X-100 (1% for 30 min at 4°C)
resulted in the solubilization of the majority of the proteins (Fig.
6D), whereas treatment with Na2CO3 resulted in the release of
some proteins (not shown). The presence of Hsp70 in ECM was
resistant to hyaluronidase treatment, suggesting that the protein is
not associated with the extracellular matrix. The total protein pattern of ECM was similar between samples isolated from control or
HS cells (Fig. 6E), which suggests that a significant difference
between ECM derived from stressed and nonstressed cells is the
presence of Hsp70. To investigate whether these ECM were
derived from the plasma membrane, control or HS cells were surface labeled using sulfo-NHS-biotin, extensively washed to remove the reagent excess, and incubated in SF conditions for 24 h.
The ECM fraction was isolated by high-speed centrifugation and
found labeled with biotin, suggesting that they were derived from
the plasma membrane of both control and HS cells. ECM were
unlikely to be derived from apoptotic cells because PS was not
detected on the surface of cells after HS and recovery. Moreover,
we did not observe any change in cellular viability during HS/
FIGURE 6. ECM derived from stressed cells contain Hsp70. HepG2
cells were maintained at 37°C (C) or heat shocked (43°C for 1.5 h), and
recovered (37°C) in SF medium. ECM were isolated from culture medium
by high-speed centrifugation as described in Materials and Methods. A,
Detection of Hsp70 by Western blotting in total cell lysates (L) or in isolated ECM derived from cells after HS and recovery for 3, 8, and 24 h. B,
Detection of Hsc70, Hsp90, and actin by Western blotting in ECM derived
from HS (24-h recovery at 37°C) or control (C) cells. C, Isolated ECM
from HS or control cells were incubated with Triton X-100 (1% for 30 min
4°C) or Na2CO3 (0.1 M at pH 11.5) and 2 mM EDTA (30 min at 4°C),
centrifuged at 100,000 ⫻ g for 1 h, and the pellet and supernatant were
analyzed for the presence of Hsp70 or GM1. D, Isolated ECM from HS or
control cells were labeled using sulfo-NHS-biotin and treated with Triton
X-100 (1% for 30 min at 4°C), centrifuged at 100,000 ⫻ g for 1 h, and the
pellet (P) and supernatant (S) were collected. E, Protein pattern of ECM
labeled with sulfo-NHS-biotin. The protein pattern in each fraction was
visualized after SDS-PAGE and transferred onto nylon membranes using
HRP-conjugated streptavidin.
recovery in comparison with control cells as assessed by the MTT
method.
The addition of yellow fluorescent protein (YFP) to the Hsp70 C
terminus abolished the insertion of Hsp70 into the plasma
membrane and its release into the extracellular medium
Our results suggest that Hsp70 is inserted into the plasma membrane with the C terminus outside the cell. A plasmid containing a quimeric Hsp70 with YFP added to the C-terminal of the
molecule (provided by Dr. H. Kampinga at the University of
Groningen, The Netherlands) was transfected into HepG2 cells.
Cells were also transfected with wild-type (WT) Hsp70. As
positive control, cells were treated with GA or subjected to
thermal stress and recovery (HS). Western blotting of lysates
obtained from transfected cells demonstrated the expression
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FIGURE 5. Hsp70 on the cell surface is resistant to Triton X-100 treatment. A, HepG2 cells were heat shocked (43°C for 1.5 h), recovered for 7 h
at 37°C, and incubated with FITC-conjugated cmHsp70.1 Ab, Alexa Fluor
532-conjugated CTX, or Alexa Fluor 592-conjugated transferrin at 4°C.
Cells were fixed with PFA and treated or not with Triton X-100 (1% for
20 min at 4°C). B, HepG2 cells after HS and recovery were incubated
with Alexa Fluor 532-conjugated CTX at 4°C for 30 min, fixed with
PFA, permeabilized with cold acetone, incubated with FITC-conjugated
cmHsp70.1 Ab at 4°C, and treated with Triton X-100 (1% for 20 min at
4°C). Nuclei were stained with DAPI. The images corresponding to
Hsp70 and GM1 were merged to show colocalization between these two
molecules. The inset shows the colocalization of Hsp70 and CTX on the
membrane.
4303
4304
EXPORT OF MEMBRANE-BOUND HSP70
FIGURE 7. The interaction of Hsp70 with membranes is affected by
addition of YFP at the C-terminal of the hsp. HepG2 cells (30% confluency) were transfected using FuGENE with either pYFP-N1-Hsp70
(YFP) encoding a YFP-Hsp70 quimera (provided by H. Kampinga, University of Groningen, The Netherlands) or pSG5–12c, a WT Hsp70
plasmid. After transfection (72 h), cells were lysed or total membrane
fraction was isolated. Lysates or total membranes were obtained from
nonstressed cells (C) as negative control or cells treated with GA or
stressed (HS) as positive controls. A, Detection of Hsp70 by Western
blotting from cell lysates (notice that Hsp70-YFP displayed a higher
m.w.). B, Detection of Hsp70-YFP or WT transgene expression in permeabilized cells by fluorescent microscopy. Inset marked by dotted line
is magnified in lower right corner inset. C, Presence of Hsp70 on the
cell surface after transfection with WT Hsp70 visualized by the Ab
against the C-terminal of the molecule (cells were incubated with the
Ab at 4° C). Inset on the left panel is displayed at high magnification on
the right panel, demonstrating the presence of WT Hsp70 on the cell
surface. D, Detection of Hsp70 by Western blotting in isolated total
membrane preparations. E, Detection of Hsp70 in ECM obtained from
cells transfected with Hsp70-YFP or Hsp70 WT constructs (72 h).
levels for YFP-Hsp70 and WT Hsp70 in comparison to Hsp70
induced by HS or GA treatment. Hsp70-YFP displayed a higher
m.w. than WT Hsp70 (Fig. 7A). In addition, the presence of
Hsp70-YFP was visualized by fluorescent microscopy (Fig.
7B). Although a robust expression level was observed for the
quimeric protein, Hsp70-YFP was not observed on the plasma
membrane (Fig. 7B). In contrast, cells transfected with WT
Hsp70 showed the presence of Hsp70 in the cytosol as well as
on the plasma membrane (Fig. 7B; see inset). The presence of
WT Hsp70 on the plasma membrane of transfected cells was
confirmed by the detection of this protein using FITC-conjugated cmHsp70.1 Ab at 4°C (Fig. 7C). Furthermore, Western
blot analysis of isolated total membrane fraction from transfected cells revealed the presence of WT Hsp70, whereas
Hsp70-YFP (higher m.w.) was not observed (Fig. 7D). Isolated
ECM from transfected cells revealed the presence of WT
Hsp70, but not YFP-Hsp70 (Fig. 7E). These observations suggest that insertion of Hsp70 into the plasma membrane requires
an intact C terminus of the molecule. In addition, insertion in
the plasma membrane may be a requirement for its release
within ECM.
ECM containing Hsp70 are capable of activating macrophages
To investigate the potential biological role of Hsp70-positive
ECM, macrophages (J744.A1) were incubated for 3 h with ECM
derived from either control or HS (24 h of recovery from the
stress) cells in SF medium. At the end of the incubation period, the
extracellular culture medium was collected for the detection of
TNF-␣. Macrophages exposed to ECM containing Hsp70 produced higher levels of TNF-␣ (7.5-fold higher) in comparison with
ECM derived from control cells. TNF-␣ production was also induced by the addition of recombinant human Hsp70 (50 ng) under
similar conditions (Fig. 8). However, TNF-␣ production by
Hsp70-positive ECM was 25.9-fold higher than the cytokine levels
after incubation with recombinant Hsp70. The amount of Hsp70
within HS-derived ECM was estimated to be ⬃5 ng by Western
blotting using recombinant protein as standards. Thus, the specific
difference in TNF-␣ production between Hsp70 within ECM and
recombinant protein is ⬃260-fold. The fact that macrophages were
incubated with ECM in SF medium reduces the possibility that the
activation of macrophages was due to contamination with endotoxin. This assumption was further supported by the observation
that boiling ECM for 30 min resulted in an ⬃84% reduction of
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 8. ECM derived from HS cells activates the production of
TNF-␣ by macrophages. Macrophages (J774; 2.5 ⫻ 105 per well) were
incubated with ECM (50 ␮l) derived from control or heat-shocked HepG2
cells in SF medium for 3 h at 37°C. At the end of the incubation period,
extracellular medium was collected and stored at ⫺20°C. Levels of TNF-␣
in the extracellular medium were measured by ELISA and normalized by
the number of viable cells in each well measured by the MTT assay. Results are presented as mean ⫾ SEM (n ⫽ 10). Statistical analysis was
performed by one-way ANOVA, followed by Newman-Keuls test.ⴱ, p ⬍
0.05 vs cells maintained in SF medium without additions; ⴱⴱ, p ⬍ 0.05 vs
cells incubated with ECM derived from control (C) or rHsp70; #, p ⬍ 0.05
vs cells incubated with rHsp70.
The Journal of Immunology
TNF-␣ production by J744 cells. To demonstrate that the biological effect of ECM containing Hsp70 is due to the presence of this
protein, cells were HS in the presence of actinomycin D, which
was washed off after 3 h of recovery from the stress. This treatment
did not result in any loss of cellular viability. However, expression
of Hsp70 was dramatically reduced in cell lysates from actinomycin D-treated cells in comparison with cells in absence of the drug
(Fig. 9A). ECM were isolated from HS cells treated or not with
actinomycin D or from nonstressed cells. ECM isolated from actinomycin D-treated HS cells showed the same level of TNF-␣
production as controls cells, which was considerably lower than
incubation with ECM derived from HS cells in absence of actinomycin D treatment (Fig. 9B).
Discussion
Hsp70 is an intracellular chaperone, which is mainly involved in
protein folding. A great body of evidence has demonstrated that
this protein could also be detected outside cells where it has the
capacity to activate the immune system (2, 5). Early assumptions
indicate that Hsp70 was released into the extracellular medium
from necrotic cells (11). Although this possibility cannot been
discarded completely, it has been demonstrated that extracellular hsps are present in the absence of cell death (12). Because
Hsp70 does not have a consensual secretory signal, the mechanism for translocation of this protein across membranes is intriguing. The earliest observation regarding the release of Hsp70
from viable cells by a nonclassical mechanism was reported by
Hightower and Guidon (23). In their study, extracellular Hsp70
was found noncovalently associated with fatty acids. More recently, it has been shown that Hsp70 could be secreted from intact
cells via exosomes (16, 17, 22, 24) or by an endo-lysosomal-dependent pathway (25). In the present study, we provide evidence
that Hsp70 is inserted into the plasma membrane before release
into the extracellular environment in membrane-associated structures from intact stressed cells. This membrane-bound Hsp70 is
capable of activating macrophages.
Previous studies from our laboratory have shown that Hsp70
and Hsc70 interact with lipid membranes (13), displaying a high
degree of specificity for the presence of PS (14). The interaction of
Hsp70 and Hsc70 with lipids is a unique characteristic of these two
proteins because other hsps, including Hsp90 and Hsp60, do not
associate with artificial membranes (A. De Maio and N. Arispe,
unpublished observations). In agreement with these observations,
prior investigations have detected Hsp70 in close proximity to cellular membranes (26, 27). Moreover, the presence of Hsp70 on the
surface of transformed cells has been well documented (17, 28).
We observed that, indeed, Hsp70 could be detected on the surface
of nonpermeabilized cells after heat shock, which is consistent
with prior observations in tumor cells (28). Our results indicate
that Hsp70 is embedded within the plasma membrane as demonstrated by selective Ab binding. These observations suggest that
Hsp70 may span the plasma membrane, leaving a small region of
the C terminus exposed outside of the cell while the N terminus is
located on the cytosolic side. This assumption is further supported
by the ability of Hsp70 to integrate into artificial lipid bilayers and
open ion conductance pathways. A similar channel activity has
been previously reported for Hsc70 (19). The C-terminal of Hsp70
(peptide binding domain) is long enough to span the typical 50-Å
width of the plasma membrane. In addition, this region presents
␤-sheet structures, which may act as centers for Hsp70 oligomerization and insertion into the lipid membrane. Other molecules
containing similar ␤-sheet structures have also been found to form
channels, including annexin, ␤-amyloid, and amylin (29 –31). The
mechanism that has been proposed to explain the selective insertion of Hsp70 into membranes assumes that Hsp70 is capable of
assembling into low-order oligomers when the protein is in excess
of polypeptide targets, such as when proteins become unfolded
after stress (14). Indeed, Hsp70 has been reported to oligomerize in
the absence of peptide targets or denatured proteins in an ATPdependent manner (32, 33). Higher-order oligomerization of proteins has been proposed to target them to the plasma membrane
before exosome budding (34). Because Hsp70 displayed a high
degree of specificity for PS (14), which is a component of the
cytosolic side of cellular membranes, we speculate that Hsp70
could translocate spontaneously from the cytosol into the plasma
membrane after oligomerization and binding to PS. Thus, Hsp70
could initially associate with PS on the cytosolic phase of the
plasma membrane and translocate within the lipid bilayer by the
spontaneous flipping of this lipid into the outside of the cell. This
process may be followed by the rapid ATP-dependent return of PS
to the inner side of the plasma membrane, leaving Hsp70 inserted
into the membrane. Elevation of surface PS is a characteristic of
apoptotic cells due to inhibition of the ATP-dependent flipase that
returns the phospholipid inside the plasma membrane. It is unlikely
that the insertion of Hsp70 into the plasma membrane is related to
the apoptotic process. In fact, we did not detect surface PS on
HepG2 cells after HS and recovery. Cell viability was not compromised under HS and recovery conditions either. Moreover,
there are no reports, to the best of our knowledge, indicating that
apoptotic cells contain a large amount of Hsp70 on the plasma
membrane.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 9. ECM derived from HS cells in the presence of actinomycin
D did not induce the production of TNF-␣ by macrophages. A, HepG2 cells
were heat shocked (HS for 1.5 h) in presence or absence of actinomycin D
(AcD; 1 ␮g/ml). Cells were allowed to recover (37°C) for 3 h in the presence or absence of actinomycin D and then the drug was removed and the
cells were allowed to recover for up to 24 h. Nonstressed cells were used
as a control (C). B, ECM were isolated from control or HS cells treated or
not with actinomycin D as described above. ECM were incubated with
macrophages (J774; 2.5 ⫻ 105 per well) in SF medium for 3 h at 37°C. At
the end of the incubation period, extracellular medium was collected and
stored at ⫺20°C. Levels of TNF-␣ in the extracellular medium were measured by ELISA and normalized by the number of viable cells in each
well measured by the MTT assay. Results are presented as mean ⫾
SEM (n ⫽ 8). Statistical analysis was performed by one-way ANOVA
followed by Newman-Keuls test. ⴱ, p ⬍ 0.05 vs cells maintained in SF
medium without additions; ⴱⴱ, p ⬍ 0.05 vs cells incubated with ECM
derived from control (C).
4305
4306
stimulated the cytosolic capacity of NK cells (17). Hsp70 has been
found in serum obtained from patients suffering from an array of
conditions, such as cancer (40, 41), diabetes (42), coronary artery
disease (43, 44), myocardial infarction (45), and trauma (46). The
presence of Hsp70 in circulation within membranes or in a free
form still needs to be established. Finally, we cannot discard the
possibility that the release of Hsp70 could be part of a mechanism
to limit the presence of Hsp70 in cells. There is evidence that
Hsp70, despite being protective in the short term, could be cytotoxic in the long term (14, 47). Indeed, the expression of Hsp70 is
tightly regulated at the level of transcription (48) and mRNA stability (49). It is possible that the release of Hsp70 upon interaction
with lipid rafts is an additional mechanism for avoiding a secondary negative effect of this protein.
In summary, we have shown that Hsp70 is present on the cell
surface. We propose that Hsp70 translocates into the plasma membrane before the release of this protein in a membrane-associated
form, which may be derived by inverse evagination, exocytosis, or
membrane shedding. Hsp70 within these membranes could act as
an immune modulator, serving as a danger signal. Thus, it could be
speculated that ECM-bound Hsp70 is involved in the activation of
the immune system to generate a systemic response to a localized
insult to avoid the propagation of the initial stress.
Acknowledgment
We thank Molly Wofford for editorial assistance.
Disclosures
The authors have no financial conflict of interest.
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Our data also showed that Hsp70 could be colocalized within
DRM depending on the presence of cholesterol, because this protein was no longer detected in these fractions after treatment with
M␤CD. Similar observations were obtained with cells expressing
Hsp70 on the surface. Hsp70 was observed to colocalize with GM1
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